Algae Balls Investigating Photosynthesis

Teacher Materials

In this lab, students will learn about photosynthesis by creating “algae balls”. They will immobilize algae in sodium alginate and measure the photosynthetic rate using a carbon dioxide indicator.

Learning Goals, Objectives, and Skills………………………………………………………………………………………………….2

Instructor Planning Guide……………………………………………………………………………………………………………………3

Instructor Preparation Guide………………………………………………………………………………………………………………6

Answers to Student Questions……………………………………………………………………………………………………….…..9

Standards Alignments……………………………………………………………………………………………………………………….11

Appendices……………………………………………………………………………………………………………………………………….12

1. Color Standards Preparations and Examples……………………………………………………………………………..122. Set-ups for Additional Independent Variables…………………………………………………………………………..143. Using Vernier Equipment and Software to Determine pH…………………………………………………………17

Last updated: 2/20/2018

Algae BallsLearning Goals

Student Learning Goals:

Students will understand the basic process of photosynthesis.

Students will understand the role of environmental factors on photosynthetic rate.

Student Learning Objectives:

Students will articulate the function of photosynthesis and identify the reactants and products of this reaction.

Students will measure the effect of light and other factors on photosynthesis.

Scientific Inquiry Skills:

Students will pose questions and form hypotheses.

Students will design and conduct scientific investigations.

Students will make measurements and record data.

Students will use mathematical operations to analyze and interpret data.

Students will generate tables and graphs to present their data.

Students will use experimental data to make conclusions about the initial question and to support or refute the stated hypothesis.

Students will follow laboratory safety rules and regulations.

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Algae BallsInstructor Planning Guide

Experimental Timing Tips:As written, the first day of this lab takes about 60 minutes. It should take the students 20-25 minutes to make the algae and water balls and then the first incubation period is 30 minutes.

If your class is less than 60 minutes, it is recommended that you break this lab into 2 days. On day 1, have the students make the algae and water balls. Have the students incubate their tubes overnight and then on day 2, students can look at the tubes and record their results.

Specialized Equipment: Equipment for culturing algae:

o Fluorescent lighto Materials for aerating algae. For example:

aquarium pump platform shaker

Ordering information:If you are planning on doing this lab with several classes it is recommended that you order algae food source, item # 142337, from Carolina Biological Supply Company. This 1-quart culture can be concentrated by centrifugation or by letting the algae settle to the bottom of the bottle for a couple of days and then gently pour the water off the top. You can also “bulk” up the culture by growing it under white fluorescent light for several weeks. Adding additional carbon dioxide by blowing into it will enhance the growth.

For one or two classes, we recommend you purchase either Chlamydomonas (#131738) or Chlorella (#152069) from Carolina.

Procedure Tips:1. Before starting the experiment, ask students to check their materials list to make sure that they

have all materials.

2. Review the use of transfer pipettes with students, show them how to transfer liquid without getting any bubbles.

3. In order to use the least number of transfer pipettes, make sure students label pipettes and beakers correctly.

4. Caution students about touching the calcium chloride solution with the tip of their alginate pipette because it may make a blockage that stops their algae mix from coming out of the pipette.

5. A convenient stopping point in the protocol is after the students have made the algae and water balls but before they add the indicator solution. You can store the algae balls for a few weeks submerged in distilled H2O in the refrigerator.

6. If you are planning to use a spectrophotometer to measure the pH change, it is recommended that you use larger reaction tubes for the experiment. Using a 5 mL tube or vial will provide enough solution to fill the cuvettes. Students will also need to make more algae balls so start them off with 1

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mL of concentrated algae and 1 mL of alginate and this will allow them to make 40-50 balls. The same change should be made in the procedure for making water balls.

Teaching Tips:1. If your algae balls are not very firm or alternatively, too firm, you may need to alter the ratio of algae

to sodium alginate. Do a test run before aliquoting the student materials. Make sure to tell the students if you change the ratio.

2. The concentration of algae in the algae balls will affect the rate of photosynthesis. Therefore, it is essential that each group is given algae from the same ‘batch’ so they can compare data.

3. There are three options to measure pH. a. The first is to compare the colors in the vials to the colors in the chart, Color Standards

and Corresponding pH Values, found in Appendix 1. b. The second is to compare your solution of interest against color standards titrated to

known pH values. The indicator is highly sensitive from pH 7.6 to 9.2. The Color Standards Preparation (Appendix 1) section of the Laboratory Setup Manual describes how to prepare these.

c. The third option, which is the most quantitative, is to measure the absorbance of your solution at 550 nm using a Vernier UV-vis spectrophotometer (Appendix 3). Absorbance at this wavelength is linear with pH in the range of 7.6 to 9.2. This allows you to generate a standard curve of absorbance vs. pH from which you can interpolate data.

4. The amount of Algae Mix (concentrated algae and alginate) that is prepared in this protocol makes enough algae balls for this experiment. If you are planning to have your students continue their investigations by designing their own experiment, they will need additional Algae Mix. There are two different ways to prepare for this:

a. Once the students have set up their first experiment, they can use the 30-minute incubation time to decide what further investigation they would like to do and prepare additional algae (and water balls if needed). Students can either make new Algae Mix or work from a stock bottle of Algae Mix.

b. Or students can reuse their algae balls from the first experiment. In order to reuse the algae balls, they need to be removed from the indicator, rinsed with tap water, and then stored in tap water at 4C until needed.

Safety Considerations: Gloves, lab coats and eye protection should be used whenever possible, as a part of

good laboratory practice.

Always wash hands thoroughly after handling biological materials or reagents.

Obtain the Material Safety Data Sheets (MSDS), available from the suppliers, for the reagents and follow all safety precautions and disposal directions as described in the MSDS.

Check with your school’s lab safety coordinator about proper disposal of all materials.

Content Information:

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Students will immobilize defined quantities of algae in gelatinous balls of calcium alginate. The balls are formed when sodium alginate is added to a solution of calcium chloride. A double replacement reaction occurs, replacing the sodium ions with calcium ions. The crosslinking that occurs stabilizes the compound, creating the balls. These balls will be immersed in solutions that allow for controlled testing of an independent variable. The pH of this solution over time serves as a proxy for photosynthetic rate. pH changes result from uptake of CO2 during photosynthesis or production of CO2 during cellular respiration, via the reaction:

CO2 (g) + H2O (l) <=> H+ (aq) + HCO3

- (aq)

Students will monitor pH changes in the reaction vials by watching the color changes of the hydrocarbonate indicator solution. The solution becomes more basic as the algae takes up the CO2, resulting in a change from yellow to purple.

An excellent reference for technical support and for ideas about additional experiments is http://www.saps.org.uk/secondary/teaching-resources/235-student-sheet-23-photosynthesis-using-algae-wrapped-in-jelly-balls. We encourage you to adapt this lab to your vision and your curricular needs once you become comfortable with the physical manipulations. For instance, it is easily modified to test the effects of different factors on cellular respiration, or to compare photosynthetic rates between different microalgae.

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Algae BallsInstructor Preparation Guide

Materials: This guide assumes 30 students, working in groups of two, for a total of 15 groups.

Materials for Teacher Advanced Preparation:

Concentrated liquid freshwater algae suspension (Chlorella or Chlamydomonas) 75 1 mL graduated transfer pipet 45 small beakers or disposable plastic cups 15 tea strainers (or 20cm x 20cm plastic screen) 90 2.0 mL (or larger) tubes 15 plastic spoons 15 microcentrifuge tube racks lamp (15 watt spiral fluorescent bulb works well) sodium alginate (powdered) calcium chloride (powdered) baking soda (sodium bicarbonate) cresol red thymol blue ethanol dH2O

Optional Supplies/equipment Platform shaker Color Standards 9 X 10 mL glass vials with lids Boric acid Sodium tetraborate decahydrate or sodium borate (Borax)

Materials for each Student Workstation: Materials for the Common Workstation: 6 graduated transfer pipettes 2 small beakers or paper cups tea strainer or mesh filter plastic spoon 6 2.0 mL microcentrifuge tubes sodium alginate calcium chloride 1X hydrocarbonate indicator solution

(orange) dH2O permanent marker

tap water color standards

Set-up Calendar:

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6 weeks before the lab*: Order algae.

4 weeks before the lab (or when the algae arrives): Set up culturing conditions for algae. The algae will grow well under white fluorescent light at

room temperature for at least 16 hours/day. If using an incandescent bulb, use a beaker of water as a heat sink so the algae does not overheat.

o This mixture will need to be aerated with an air stone and pump. An alternative to the pump would be continuous shaking. The culture should be a dark green (think pea soup!)

o For additional information see: http://resources.wardsci.com/livecare/algae-cyanobacteria/

3-7 days before the lab:

Copy ‘Color Standards Charts’ for student work stations (page 13) or prepare liquid Color Standards (Appendix 1)

Copy student labs. Prepare the following three solutions:

Sodium Alginate (2%) 1. Add 2 g sodium alginate to 100 mL dH2O and shake until dissolved. The alginate

may not go into solution initially but if left over night at room temperature, it will go into solution.

2. Note, heating this solution is NOT recommended. Since this is a natural product, the viscosity may vary from batch to batch. The alginate should be fluid enough to pour slowly. If it is too viscous, add dH2O to thin it.

3. Store covered in refrigerator.

4. Let warm to room temperature before using. Solution will last for several weeks if stored at 4C.

Calcium Chloride (2%): 1. Add 10 g calcium chloride to 500 mL dH2O and stir until dissolved.

2. Dispense 20 mL into 15 50 mL conical tubes and store at 4C until lab day.

Hydrocarbonate indicator:1. Dissolve 0.1 g cresol red and 0.2 g thymol blue in 20 mL of ethanol.

2. Dissolve 0.85 g of baking soda in 200 mL dH2O.

3. Add the cresol red/thymol blue/ethanol solution to the baking soda solution and then bring the volume up to 1000 mL with dH2O.

4. This is the 10X stock solution. To use, dilute to 1X.

5. If the diluted 1X indicator is blue or purple, use a straw to blow CO2 into it until it is orange.

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1 day before lab:

Prepare concentrated algae for student stations. 1. Concentrate the algae:

Depending on how much time you have, there are two ways to concentrate the algae. Both involve concentrating the algae and then re-suspending it to get the volume needed.

a) let the algae settle at the bottom of the bottle overnight and then slowly pour off the liquid—leaving the concentrated algae solution at bottom. Save both the algae and the decanted liquid.

b) add 45 mL of algae solution to 50 mL conicals and centrifuge at low speed for 5 minutes. Pour off and save the supernatant.

2. Make the final algae solution:

Each pair of students will need 0.75 mL of algae solution. Using the concentrate from either method above, bring the volume up to 20 mL (if you have 15 pairs). The supernatant or the decanted liquid should be used to re-suspend the algae. This solution should be stored at room temperature until the lab period. Aliquot 0.75 mL of algae into 15 tubes.

Set up student lab stations. Set out the color standards (the chart or standards that you made).

Algae Balls8

Answers to Student Questions

Protocol-embedded:

p. 2:

The biological process is photosynthesis. The energy for photosynthesis comes from sunlight. The carbon for production of biofuel comes from CO2. What do you already know? sample answers

o Photosynthesis fixes CO2 into glucose.o Photosynthesis uses chlorophyll.o Photosynthesis has light dependent and light independent reactions.o Photosynthesis requires ATP and NADPH.o Green plants and other primary producers are the only organisms the can

photosynthesize, etc.

p. 3:

p. 4:

The tube on the left, the one that is more yellow, has more H+ ions. Not much photosynthesis has occurred, so there is lots of carbon dioxide dissolved in water,

which produces carbonic acid, H2CO3, and lowers the pH.

Pre-lab:

1. Photosynthesis uses energy from the sun to drive the reaction: CO2 + 6H2O C6H12O6 + 6O2

2. The tube containing the water balls was our control. An experimental control increases the reliability of the results by making sure that nothing other than the single independent variable is being tested.

3. The hydrocarbonate indicator changes color as the pH changes. As the algae performs photosynthesis, the carbon dioxide concentration decreases making the solution more basic.

4. The water balls should not have any photosynthetic organisms, so the reaction above should not occur, and we should not see any change in pH. If something else is going on in the experiment, we would be able to detect that with the water ball control.

5. Table 2: Result Predications

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TimePredictions

Algae/ Light Algae /Dark Water/ Light Water/ Dark

30 min

Some of the CO2 will be converted to glucose, which will decrease the amount of carbonic acid, and thus the pH will increase, and the color of the indicator solution will begin to change from yellow to orange or red.

Light is a component of

the photosynthetic reaction and it cannot occur without. The

amount of carbonic acid will stay the

same, no color change.

The CO2 will NOT convert to glucose, the amount of

carbonic acid will remain the same, and thus the pH will remain the same and there

will be no color change.24 hours

Most of the CO2 will be converted to glucose and the color of the indicator solution will be purple.

6. The dyes in the indicator are sensitive to pH. When CO2 is present, the level of carbonic acid is high, and the pH is low. Photosynthesis consumes CO2, so a color change associated with a decrease in CO2 can be detected.

Post-Lab and Analysis:

1. (Sample answer) The results from the algae ball tube and the control tube matched my prediction. The water ball tube did not change color, but the algae ball tube became purple with time.

2. Answers will vary depending upon the measurement tools used.

3. Answers will vary depending upon the design of each individual student’s experiments.

4. (Sample answer) If the algae was boiled before making algae balls, then I would expect the cells to be dead and unable to photosynthesize. If the algae cells were unable to photosynthesize, then the algae ball and water ball tubes’ indicator should be the same color.

5. (Sample answer) If the water used to make the water balls was contaminated with bacteria, the results would probably be the same as the uncontaminated control. Bacteria do not photosynthesize so the CO2 that is present will remain. When given glucose or another carbon source to consume, bacteria do respire, which produces CO2. But, in the absence of glucose or another carbon source to consume the bacterial contaminated water balls would probably behave much like the normal control water balls.

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Algae BallsStandards Alignments

MA Science and Technology/Engineering Curriculum Framework (2006)

Biology

1.2 Describe the basic molecular structures and primary functions of the four major categories of organic molecules (carbohydrates, lipids, proteins, nucleic acids).

2.4 Identify the reactants, products, and basic purposes of photosynthesis and cellular respiration. Explain the interrelated nature of photosynthesis and cellular respiration in the cells of photosynthetic organisms.

2.5 Explain the important role that ATP serves in metabolism.

Chemistry

7.5 Identify the factors that affect the rate of a chemical reaction (temperature, mixing, concentration, particle size, surface area, catalyst).

Scientific Inquiry Skills

SIS1. Make observations, raise questions, and formulate hypotheses.

SIS2. Design and conduct scientific investigations.

SIS3. Analyze and interpret results of scientific investigations.

SIS4. Communicate and apply the results of scientific investigations.

Mathematical Skills

Construct and use tables and graphs to interpret data sets.

Solve simple algebraic expressions.

Perform basic statistical procedures to analyze the center and spread of data.

Measure with accuracy and precision (e.g., length, volume, mass, temperature, time)

Use common prefixes such as milli-, centi-, and kilo-.

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Appendix 1Color Standards Preparation and Recipes (10 mL and 2 mL samples)

You can either print the image below for students to use as their color standards or you can prepare your own color standards using the directions below the image. There are two sets of instructions: 1 is to make 10 mL color standards, and 2 is to make 2 mL color standards.

Color Standards and Corresponding pH Values

Photo by Debbie Eldridge. Copyright Science & Plants for Schools (www.saps.org.uk). Photosynthesis with Algal Balls: Technical notes (Revised 2012).

Recipe makes up to 10 sets of 10 mL color standards.

1. Dissolve 3.1 g boric acid in 240 mL dH2O and stir until dissolved. Bring volume to 250 mL.

2. Dissolve 2.9 g of sodium tetraborate decahydrate in 140 mL of dH2O and stir until dissolved. Bring volume to 150 mL.

3. Use a clean container to make each pH standard to avoid contamination. For each pH color standard, add the boric acid solution and the borax solution in the amounts in the table below to a small flask or beaker and bring the solution to 100 mL final volume with dH2O. These are now your stock bottles.

pH Color Standards – Recipe Chart

pH 7.6 7.8 8.0 8.3 8.4 8.6 8.8 9.0 9.2

Boric Acid 25 mL 25 mL 25 mL 25 mL 25 mL 25 mL 25 mL 25 mL 25 mL

Borax 1.0 mL 1.55 mL 2.45 mL 3.8 mL 5.7 mL 8.7 mL 15.0 mL 29.5 mL 57.5 mL

Add enough dH2O to bring the total volume in each container to 100 mL

4. Label one of each of 9 X 10 mL clean glass vials with one of the following pH values: pH 7.6, pH 7.8, pH 8.0, pH 8.2, pH 8.4, pH 8.6, pH 8.8, pH 9.0, pH 9.2. Repeat to generate the desired number of standards sets.

5. Dispense 9 mL of each pH color standard into the corresponding 10 mL glass vial. Store at room temperature until ready to use. Four to six sets of standards are sufficient for a class of 30 students (~ 1 set of standards/3 groups). Repeat to generate the desired number of standards sets.

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pH 7.6 pH 7.8 pH 8.0 pH 8.2 pH 8.4 pH 8.6 pH 8.8 pH 9.0 pH 9.2

6. Add 1 mL of 10X hydrocarbonate indicator solution to each color standard vial. Refer to Set-Up Manual for instructions.

Recipe makes 1 set of 2 mL color standards.

1. Dissolve 0.31 g boric acid in 24 mL dH2O and stir until dissolved. Bring volume to 25 mL.

2. Dissolve 0.29 g of sodium tetraborate decahydrate (borax) in 14 mL of dH2O and stir until dissolved. Bring volume to 15 mL.

3. For each pH color standard, add the boric acid solution, the borax solution and the 10X hydrocarbon indicator in the amounts in the table below to a 2 mL tube and bring the solution to 2 mL final volume with dH2O.

pH Color Standards – Recipe Chart (2.0 mL tubes)

pH 7.6 7.8 8.0 8.3 8.4 8.6 8.8 9.0 9.2

Boric Acid 500 μL 500 μL 500 μL 500 μL 500 μL 500 μL 500 μL 500 μL 500 μL

Borax 20 μL 31 μL 49 μL 72 μL 114 μL 174 μL 300 μL 590 μL 1150 μL

10X indicator 200 μL 200 μL 200 μL 200 μL 200 μL 200 μL 200 μL 200 μL 200 μL

Add enough dH2O to bring the total volume in each container to 2 mL

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Appendix 2: Set-ups for Additional Independent Variables

Although we offer the following guidelines for obtaining robust results, factors such as ambient temperature and the inherent activity of your microalgae culture will affect photosynthetic rate. We encourage your students to be flexible and creative in adjusting various features of the setup.

The algae vial should be placed close to a 60W incandescent equivalent (or brighter) light bulb in order to visualize changes within a class period. With the exception of the investigation of different light sources, we recommend compact fluorescent bulbs because they produce less heat.

A. Color filters or light bulbs of different colors provide a simple but powerful illustration of the absorption properties of photosynthetic pigments. Each filter transmits the color that it appears to our eyes and absorbs other visible wavelengths to varying degrees. Ask your students to consider the absorption spectrum of chlorophyll and minor photosynthetic pigments. Which filter should allow the greatest activity? the least?

1. Gather red, blue, green, and transparent color filters. Which is your experimental control?2. Add 35 algae balls to each of four vials and 35 H2O/alginate balls to another vial. With which

sample will you compare your H2O control?3. Prepare 20 mL of 1X hydrocarbonate indicator solution. Dispense 3.0 mL to each vial. Invert the

vial gently and allow the solution to equilibrate for 3 minutes.4. Determine the initial pH:

a. If comparing against color standards, estimate pH to the nearest tenth.b. If using the UV-vis, transfer 1.5 mL from each vial to a cuvette for an initial absorbance

reading. Be sure to keep track of the sample to which each cuvette corresponds. Then re-pour the solution from the cuvette back into its corresponding sample vial.

5. For each filter setup, place the vial at an angle into a paper cup. Sit the filter over the mouth of the cup.

6. Shine a CFL bulb over all setups. Be sure that all vials are equidistant from the light source.7. Record the final pH:

a. If comparing against color standards, estimate pH to the nearest tenth.b. If using the UV-vis, remove 1.5 mL of the indicator solution from each vial and transfer

to a cuvette. Be sure to keep track of which sample each cuvette corresponds to. Record the absorbance of each sample.

Sample abs at time 0 (absinitial) abs at 30 minutes (absfinal)H2O/alginate controltransparent filterred filter/lightgreen filter/lightblue filter/light

B. Different light sources highlight other factors that affect photosynthesis when the overall brightness remains constant. Can algae detect differences between light emitted by LED vs. CFL bulbs? What is a side effect of incandescent bulbs?

1. Gather LED, CFL, and incandescent bulbs of 1200 lumens.2. To 18 mL distilled H2O, add 2 mL 10X hydrocarbonate indicator solution. 3. Label each of three vials LED, CFL, and incandescent. Add 35 algae balls to each. 4. Label another vial H2O/alginate. Add 35 H2O/alginate balls. Decide on which light source you

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would like to test this control.5. Dispense 3.0 mL 1X indicator solution to each. Allow to equilibrate for 3 minutes.6. Determine the initial pH:

a. If comparing against color standards, estimate pH to the nearest tenth.b. If using the UV-vis, transfer 1.5 mL from each vial to a cuvette for an initial absorbance

reading. Be sure to keep track of the sample to which each cuvette corresponds. Then re-pour the solution from the cuvette back into its corresponding sample vial.

7. Place all vial at the same distance from their respective light sources for 30 minutes. To reduce contaminant light, consider placing cardboard barriers between the setups.

8. Record the final pH:a. If comparing against color standards, estimate pH to the nearest tenth.b. If using the UV-vis, remove 1.5 mL of the indicator solution from each vial and transfer

to a cuvette. Be sure to keep track of which sample each cuvette corresponds to. Record the absorbance of each sample.

Sample abs at time 0 (absinitial) abs at 30 minutes (absfinal)H2O/alginate controlLEDCFLincandescent

C. External carbon sources test the ability of carbohydrates to feedback inhibit enzymes required for photosynthesis. The glyceraldehyde-3-phosphate produced in the Calvin-Benson cycle has two fates: conversion into starch or into the disaccharide sucrose, which subsequently is hydrolyzed into glucose and fructose. Ask your students to hypothesize how external carbon sources affect photosynthesis rate. They can compare equal concentrations of two different sugars, such as sucrose and glucose, or varying concentrations of a single sugar. The following protocol was optimized for the latter investigation, but can be modified to test equimolar glucose and sucrose.

1. Prepare 9 mL of 0.5M glucose and 9 mL of 0.1M glucose. Add 1 mL 10X hydrocarbonate indicator solution to each.

2. To 18 mL distilled H2O, add 2 mL 10X hydrocarbonate indicator solution. 3. Label each of three vials –glucose, 0.1M glucose, and 0.5M glucose. Add 35 algae balls to each.

Add 3.0 mL 1X indicator solution to the –glucose vial and 3.0 mL of the corresponding solutions into the glucose vials.

4. Add 35 H2O/alginate balls to a fourth vial. Label it H2O/alginate. Dispense 3.0 mL 1X indicator solution into this vial.

5. Invert all vials gently and allow to equilibrate for 3 minutes.6. Determine the initial pH:

a. If comparing against color standards, estimate pH to the nearest tenth.b. If using the UV-vis, transfer 1.5 mL from each vial to a cuvette for an initial absorbance

reading. Be sure to keep track of the sample to which each cuvette corresponds. Then re-pour the solution from the cuvette back into its corresponding sample vial.

7. Place all vial equidistant from the light source for 30 minutes.8. Record the final pH:

a. If comparing against color standards, estimate pH to the nearest tenth.b. If using the UV-vis, remove 1.5 mL of the indicator solution from each vial and transfer

to a cuvette. Be sure to keep track of which sample each cuvette corresponds to. Record the absorbance of each sample.

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Sample abs at time 0 (absinitial) abs at 30 minutes (absfinal)H2O/alginate control- glucose algae control0.5 M glucose0.1 M glucose

D. The intensity of light opens a deeper consideration of the properties of chlorophyll and can also be well quantified. Ask your students to hypothesize how photosynthetic rate changes with intensity. Have them apply the inverse square law to calculate the intensity at various distances from the light source. Will the brightest light necessarily induce the most photosynthesis? Because chlorophyll is a dye with electrons that can be promoted to higher energy states, it may react with other molecules in the cell or else absorb photons in a way that causes permanent structural damage. This phenomenon is called photo bleaching. At the other extreme, what is the lowest level of light that can still support photosynthesis?

1. To 18 mL distilled H2O, add 2 mL 10X hydrocarbonate indicator solution. 2. Label each of four vials 5, 10, 20, and 30 cm. Add 35 algae balls to each. Label a fifth vial

H2O/alginate. Decide where you will place it and label it accordingly. Add 35 H2O balls.3. Dispense 3.0 mL of 1X indicator solution to each vial and invert gently. Allow to equilibrate for 3

minutes.4. Determine the initial pH:

a. If comparing against color standards, estimate pH to the nearest tenth.b. If using the UV-vis, transfer 1.5 mL from each vial to a cuvette for an initial absorbance

reading. Be sure to keep track of the sample to which each cuvette corresponds. Then re-pour the solution from the cuvette back into its corresponding sample vial.

5. Place all vial equidistant from the light source for 30 minutes.6. Record the final pH:

a. If comparing against color standards, estimate pH to the nearest tenth.b. If using the UV-vis, remove 1.5 mL of the indicator solution from each vial and transfer

to a cuvette. Be sure to keep track of which sample each cuvette corresponds to. Record the absorbance of each sample.

Sample Intensity of light (W) abs at time 0 (absinitial) abs at 30 minutes (absfinal)H2O/alginate control5 cm 10 cm20 cm30 cm

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Appendix 3:Using Vernier equipment and software to measure pH

Measuring pH (50 minutes) Students will measure the pH of their reaction solutions immediately after adding the balls and

again 30 minutes later. In the meantime, they will work in groups to generate standard curves of absorbance vs. pH.

You can connect the Vernier UV-vis spectrophotometer either to a LabQuest device or to a computer with LoggerPro. Below are step-by-step directions for how to configure and take measurements with either. We also explain how to generate and use a standard curve.

With a computer and LoggerPro software:1. Plug in the UV-vis to a USB port on your computer.2. Open LoggerPro. The software will automatically recognize the UV-vis (you will see a rainbow

spectrum on the main screen).3. You will first need to calibrate the UV-vis before your students begin taking measurements. In

LoggerPro, go to Experiment -> Calibrate -> Spectrometer: 1. The “Calibrate Spectrometer” dialog box will appear. Allow 90 seconds for the lamp to warm up.

4. Fill a clean cuvette with 1.5 mL of distilled H2O. Insert the cuvette into the UV-vis so that the clear panel faces the arrow. Make sure the water level is visible above the slot. Press the Finish Calibration button; wait for it to turn grey, which signals that the machine is done calibrating. Press OK.

a. Note: The dialog box prompts you to insert a blank cuvette. You should calibrate with a cuvette filled with distilled H2O.

5. To take absorbance measurements at a single wavelength, press the button. Under Collection Mode, select the Absorbance vs. Concentration ratio button. Under Column Name, Short Name, and Units, type “pH.” Click the arrow of the drop-down menu and select Individual Wavelengths. There will be a list of discrete wavelengths in the middle of the dialog box. Press Clear Selection, then select 550.3 nm. The right of the Clear Selection button should read “1 selected wavelengths.” Press OK.

6. A real-time panel of the absorbance reading should appear in the lower left panel. Immediately above it, there will be a two-column data table of pH as the left header and Abs-550.3 as the right header. The empty plot in the main screen should have “Absorbance at 550.3 nm” as the y-axis and “pH (pH)” as the x-axis.

7. You are now ready to collect your t0 data point. Dry off the outside of your sample cuvette and place it in the slot, ensuring that the clear panel faces the arrow. The lower left box will report the absorbance value.

With a LabQuest device:1. Press the red power button to turn on the device.2. Plug the UV-vis into the USB port on the device. The screen should read “USB: Abs.”3. Go to Sensors -> Calibrate -> USB: Spectrometer. Wait 90 seconds for the lamp to warm up.4. Fill a clean cuvette with 1.5 mL distilled H2O and insert it into the slot. Press the Finish

Calibration button. Once the screen reads “Calibration completed,” press OK. You should be back at the Meter screen.

a. Note: The interface prompts you to insert a blank cuvette. You should calibrate with a cuvette filled with distilled H2O.

5. To take absorbance measurements at a single wavelength, tap “Mode” to configure the instrument. At the top of the new screen, there will be a drop-down menu next to “Mode:.”

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Select “Events with Entry.”6. Next to “Name” and “Units,” enter “pH.” Tap OK to return to the Meter Screen.7. The screen should now read “USB: Abs @ 0 nm.” Tap the red box and select “Change

Wavelength…”8. Next to “Selected Wavelength:,” enter 550. Deselect the “Report average of wavelength band”

box. Tap OK. The red box will now display the real-time absorbance reading. 9. You are now ready to collect your t0 data point. Wipe down your sample cuvette and place it in

the slot, ensuring that the clear panel faces the arrow. Record the absorbance value.

Generating a standard curve A standard curve is a plot of absorbance versus the known pH values of your color standards. Its

purposes are:o 1) To reveal the range of pH values in which absorbance varies linearly with pH. In this

range, one is confident that changes in absorbance accurately reflect changes in pH. o 2) To allow one to calculate pH from absorbance in this range. You will be able to fit a

line of the form y = ax + b, where y is the absorbance and x is the pH. By substituting y with the absorbance of a sample, you can determine its pH by solving for x.

If many groups are sharing one UV-vis, it is recommended that they manually record absorbance values and generate linear fits using Excel rather than storing data on the device.

With Excel: 1. Connect the UV-vis either to a computer or to a LabQuest device. Calibrate and set up the

instrument as described above. 2. Fill a clean cuvette with 1.5 mL of the pH 7.6 color standard. Manually record the absorbance in

the following data table:

pH 7.6 7.8 8.0 8.2 8.4 8.6 8.8 9.0 9.2Abs (550 nm)

3. Using a clean cuvette each time, repeat step 2 for each of the color standards until your table is complete.

4. Enter the data into Excel to fit a linear regression.

With LoggerPro:1. Calibrate and set up the UV-vis as described above.

2. Press the button at the top of the screen. Fill a clean cuvette with 1.5 mL of the pH 7.6 color standard and insert into the UV-vis. The real-time absorbance reading will be displayed in the lower

left corner. Once it stabilizes, press . You will be prompted to enter the pH value. Enter 7.6.3. Using a clean cuvette each time, repeat step 2 for each color standard. Enter the corresponding pH value

when prompted. The raw data will appear in the table on the left.

4. Once you have measured all standards, press . Go to Analyze -> Linear Fit. The program will implement a linear regression. To adjust the axes, go to Options -> Graph Options -> Axes Options. “Top” and “bottom” correspond to minimum and maximum values.

With LabQuest:5. Calibrate and set up the UV-vis as described above. 6. Press the green play button at the bottom left of the Meter screen. The screen should now

display a plot with Abs @ 550 nm on the y axis and pH (pH) on the x-axis. 7. Fill a clean cuvette with 1.5 mL of the pH 7.6 color standard and insert into the UV-vis. The real-

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time absorbance reading will be displayed on the right under Abs @ 550 nm. Once it stabilizes, tap the Keep button in the bottom left corner. Enter 7.6 as the pH value and press OK.

8. Using a clean cuvette each time, repeat step 3 for each color standard. Enter the corresponding pH values.

9. Once you have finished measuring all standards, press the red stop button in the lower left corner.

10. To fit a line, go to Analyze -> Curve Fit -> Abs @ 550 nm. Under the Fit Equation drop down menu, select linear. The coefficients of the equation y = mx + b should be displayed. Tap OK.

11. For the purposes of the lab report, students can either configure the device to email the data to themselves or copy it manually to process in Excel. For the latter option, click the X|Y icon to view the raw data.

12. Sample standard curve (generated in LoggerPro):

For sample data tables and examples of how to use the standard curve, see the section following “Reaction setups for each independent variable.”

Sample Data Analysis:

The following example illustrates how to calculate pH from absorbance. From the sample standard curve above of absorbance vs. pH,

abs = 0.76 (pH) – 5.61

for absorbance values between 0.24 and 1.4.

If an absorbance measurement equals 0.84,

0.84 = 0.76 (pH) – 5.61

pH = (0.84 + 5.61) / 0.76 = 8.5

Students should then be able to complete the following table:

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Sample pH at time 0 (pHinitial) pH at 30 minutes (pHfinal) ΔpH (pHfinal – pHinitial)H2O/alginate control

algae controlindependent variable – instance 1independent variable – instance 2

A concise visual representation of such data is a bar graph of ΔpH vs. the independent variable. For instance, the experiment with exogenous glucose may look like the following:

H2O/alginate algae without glucose

algae in 0.1 M glucose

algae in 0.5 M glucose

0.00.20.40.60.81.01.21.4

Effect of glucose on photosyntheticreactions

condition

pH c

hang

e af

ter 3

0 m

inut

es (f

inal

- in

itial

)

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